Optical reporter compositions

ABSTRACT

This invention provides compositions that have a light emitting reporter linked to biomolecules, preferably, nucleotide oligomers. The light reporter particles are silylated and functionalized to produce a coated light reporter particle, prior to covalently linking the biomolecules to the light reporter particle. The light reporter particles of the invention can be excited by a light excitation source such as UV or IR light, and when the biomolecule is DNA, the attached DNA molecule(s) are detectable by amplification techniques such as PCR.

CROSS REFERENCE

This application is a continuation patent application of U.S. Ser. No.11/954,030 filed on Dec. 11, 2007, which is a continuation-in-part ofpatent application Ser. No. 11/437,265 having a filing date of May 19,2006 which claims the benefit of provisional patent application No.60/682,976 filed on May 20, 2005; U.S. Ser. No. 11/954,030 is also acontinuation-in-part of patent application Ser. No. 10/825,968 having afiling date of Apr. 15, 2004 which claims the benefit of provisionalpatent application No. 60/463,215 filed on Apr. 16, 2003; Ser. No.11/954,030 also claims the benefit of provisional patent application60/874,425 having a filing date of Dec. 12, 2006; Ser. No. 11/954,030also claims the benefit of provisional patent application Nos.60/877,875 and 60/877,869 each having a filing date of Dec. 29, 2006;each of these patent applications being hereby incorporated by referenceincluding the six co-filed patent applications having Ser. Nos.11/954,009; 11/954,030; 11/954,038; 11/954,044; 11/954,051 and11/954,055 each filed on Dec. 11, 2007; now issued as U.S. Pat. Nos.8,124,333; 8,372,648; 8,426,216; 8,415,164; 8,415,165 and 8,420,400;respectively.

FIELD

This invention pertains to compositions comprising optical reporters,and more specifically up-converting phosphors (UCP) that are linked tonucleic acids of detectable sequence.

BACKGROUND

Optical reporters such as fluorescent dyes and upconverting phosphorshave been used vigorously to detect the presence of biomolecules such asproteins and DNA molecules. Up-converting phosphor particles (UCP) havebeen encapsulated in transparent polyacrylate micro-carriers which canform covalent bonds with protein sensors via functional groups asdescribed in U.S. Pat. No. 5,132,242. Silylation techniques have alsobeen utilized for binding antibodies to UCPs. Biotin/avidin binding hasbeen utilized to detect biotinylated deoxyribonucleic acid (DNA) by UCPparticles with avidin molecules on their surfaces. Hapten/digoxigeninsandwich technology has also been used to indirectly detect the presenceof DNA with modified UCP particles.

Heretofore, the covalent binding of UCP particles to DNA molecules ofdetectable sequence has not been known. Such particles would be usefulfor labeling and identification of articles and materials and as probesfor characterizing DNA. There is accordingly a need for compoundscompositions comprising an up-converting phosphor linked to asequenceable DNA molecule. The present invention satisfies these needsas well as others.

SUMMARY

This invention relates primarily to the compositions which linkbiomolecules to visual or machine detectable reporters. Reporters can beeasily detected by using a high energy light source for excitation, andthe location of labeled biomolecules is identified by the presence of areporter. Nevertheless, after the location of biomolecules, there isoften a need to analyze biomolecules further, particularly when thebiomolecule is a DNA molecule, by PCR or sequence analysis techniques.

A composition of the formula I:(cOpR)-[L-(NA)]_(m)  I

-   -   wherein: m is an integer greater than 1, (cOpR) is a coated        optical reporter particle, (NA) is a nucleic acid oligomer of        detectable sequence and L is a linking group covalently bound to        the coated optical reporter particle and to the nucleic acid        oligomer.

The (cOpR) of the composition may comprise an upconverting phosphor(UCP) material biomolecule is a DNA molecule, by PCR or sequenceanalysis techniques.

In most embodiments, the cOpR of the composition of the invention iscoated with silica.

In some embodiments where the compositions are coated with silica, thesilica comprises at least one Si—O bond.

The (NA) of the composition may be a single or double stranded DNAmolecule having a length of between about 40 base pairs and about 1000base pairs.

The linker or L of the composition may comprise an alkylene moietyhaving a first end covalently bound to the coated optical reporterparticle and a second end covalently bound to the nucleic acid oligomer.

Where the composition comprises a (UCP), in certain embodiments, the(UCP) is an upconverting phosphor particle of the formula:Y_(x)Yb_(y)Er_(z)O₂S; orNa(Y_(x)Yb_(y)Er_(z))F₄;

wherein:

-   -   x is from about 0.6 to about 0.95;    -   y is from about 0.05 to about 0.35; and    -   z is from about 0.1 to about 0.001.

In other embodiments, the linker L of compositions having the formula ofI: (cOpR)-[L-(NA)]_(m), L has the formula:-A-R¹—B—

-   -   where R¹ is C₂₋₈alkylene, -A- is a group covalently bonded to        the surface of the coated optical reporter and —B— is a group        covalently bonded to the 3′ or 5′ end of the nucleic acid        oligomer.

In other embodiments, a composition of the invention has the formula:(UCP)-[A-R¹—B-(DNA)]_(m)

-   -   where m is an integer greater than 1; UCP is an upconverting        phosphor particle; DNA is a single or double stranded        deoxyribonucleic acid oligomer; R¹ is C₂₋₈alkylene; -A- is a        group capable of covalently bonding to the surface of the        upconverting phosphor particle and —B— is a group capable of        bonding to the 3′ or 5′ end of the deoxyribonucleic acid        oligomer.

All patents and publications identified herein are incorporated hereinby reference in their entirety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an FTIR Spectrum of a silinated coated optical reporterparticle in accordance of the methods of the invention.

FIG. 2 is a plot of a real-time PCR results for a composition of theinvention, comprising an optical reporter linked to a sequenceable DNAmolecule.

DESCRIPTION

Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. It must be noted that, as used in the specification and theappended claims, the singular forms “a”, “an,” and “the” include pluralreferents unless the context clearly dictates otherwise.

“Agonist” refers to a compound that enhances the activity of anothercompound or receptor site.

“Alkyl” means the monovalent linear or branched saturated hydrocarbonmoiety, consisting solely of carbon and hydrogen atoms, having from oneto twelve carbon atoms. “Lower alkyl” refers to an alkyl group of one tosix carbon atoms, i.e. C₁-C₆alkyl. Examples of alkyl groups include, butare not limited to, methyl, ethyl, propyl, isopropyl, isobutyl,sec-butyl, tert-butyl, pentyl, n-hexyl, octyl, dodecyl, and the like.

“Alkenyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one double bond, e.g., ethenyl,propenyl, and the like.

“Alkynyl” means a linear monovalent hydrocarbon radical of two to sixcarbon atoms or a branched monovalent hydrocarbon radical of three tosix carbon atoms, containing at least one triple bond, e.g., ethynyl,propynyl, and the like.

“Alkylene” means a linear saturated divalent hydrocarbon radical of oneto six carbon atoms or a branched saturated divalent hydrocarbon radicalof three to six carbon atoms, e.g., methylene, ethylene,2,2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene,and the like.

“Alkoxy” and “alkyloxy”, which may be used interchangeably, mean amoiety of the formula —OR, wherein R is an alkyl moiety as definedherein. Examples of alkoxy moieties include, but are not limited to,methoxy, ethoxy, isopropoxy, and the like.

“Alkoxyalkyl” means a moiety of the formula R^(a)—O—R^(b)—, where R^(a)is alkyl and R^(b) is alkylene as defined herein. Exemplary alkoxyalkylgroups include, by way of example, 2-methoxyethyl, 3-methoxypropyl,1-methyl-2-methoxyethyl, 1-(2-methoxyethyl)-3-methoxypropyl, and1-(2-methoxyethyl)-3-methoxypropyl.

“Alkylcarbonyl” means a moiety of the formula —R′—″, where R′ is oxo andR″ is alkyl as defined herein.

“Alkylsulfonyl” means a moiety of the formula —R′—R″, where R′ is —SO₂—and R″ is alkyl as defined herein.

“Alkylsulfonylalkyl” means a moiety of the formula —R′—R″—R′″ where R′is alkylene, R″ is —SO₂— and R′″ is alkyl as defined herein.

“Alkylamino means a moiety of the formula —NR—R′ wherein R is hydrogenor alkyl and R′ is alkyl as defined herein.

Alkoxyamino” means a moiety of the formula —NR—OR′ wherein R is hydrogenor alkyl and R′ is alkyl as defined herein.

“Alkylsulfanyl” means a moiety of the formula —SR wherein R is alkyl asdefined herein.

“Aminoalkyl” means a group —R—R′ wherein R′ is amino and R is alkyleneas defined herein. “Aminoalkyl” includes aminomethyl, aminoethyl,1-aminopropyl, 2-aminopropyl, and the like. The amino moiety of“aminoalkyl” may be substituted once or twice with alkyl to provide“alkylaminoalkyl” and “dialkylaminoalkyl” respectively.“Alkylaminoalkyl” includes methylaminomethyl, methylaminoethyl,methylaminopropyl, ethylaminoethyl and the like. “Dialkylaminoalkyl”includes dimethylaminomethyl, dimethylaminoethyl, dimethylaminopropyl,N-methyl-N-ethylaminoethyl, and the like.

“Aminoalkoxy” means a group —OR—R′ wherein R′ is amino and R is alkyleneas defined herein.

“Alkylsulfonylamido” means a moiety of the formula —NR′SO₂—R wherein Ris alkyl and R′ is hydrogen or alkyl.

“Aminocarbonyloxyalkyl” or “carbamylalkyl” means a group of the formula—R—O—C(O)—NR′R″ wherein R is alkylene and R′, R″ each independently ishydrogen or alkyl as defined herein.

“Antagonist” refers to a compound that diminishes or prevents the actionof another compound or receptor site.

“Aryl” means a monovalent cyclic aromatic hydrocarbon moiety consistingof a mono-, bi- or tricyclic aromatic ring. The aryl group can beoptionally substituted as defined herein. Examples of aryl moietiesinclude, but are not limited to, phenyl, naphthyl, phenanthryl,fluorenyl, indenyl, pentalenyl, azulenyl, oxydiphenyl, biphenyl,methylenediphenyl, aminodiphenyl, diphenylsulfidyl, diphenylsulfonyl,diphenylisopropylidenyl, benzodioxanyl, benzofuranyl, benzodioxylyl,benzopyranyl, benzoxazinyl, benzoxazinonyl, benzopiperadinyl,benzopiperazinyl, benzopyrrolidinyl, benzomorpholinyl,methylenedioxyphenyl, ethylenedioxyphenyl, and the like, includingpartially hydrogenated derivatives thereof, each being optionallysubstituted.

“Arylalkyl” and “Aralkyl”, which may be used interchangeably, mean aradical-R^(a)R^(b) where R^(a) is an alkylene group and R^(b) is an arylgroup as defined herein; e.g., phenylalkyls such as benzyl, phenylethyl,3-(3-chlorophenyl)-2-methylpentyl, and the like are examples ofarylalkyl.

“Arylsulfonyl means a group of the formula —SO₂—R wherein R is aryl asdefined herein.

“Aryloxy” means a group of the formula —O—R wherein R is aryl as definedherein.

“Cyanoalkyl” means a moiety of the formula —R′—R″, where R′ is alkyleneas defined herein and R″ is cyano or nitrile.

“Cycloalkyl” means a monovalent saturated carbocyclic moiety consistingof mono- or bicyclic rings. Cycloalkyl can optionally be substitutedwith one or more substituents, wherein each substituent is independentlyhydroxy, alkyl, alkoxy, halo, haloalkyl, amino, monoalkylamino, ordialkylamino, unless otherwise specifically indicated. Examples ofcycloalkyl moieties include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like,including partially unsaturated derivatives thereof.

“Cycloalkylalkyl” means a moiety of the formula —R′—R″, where R′ isalkylene and R″ is cycloalkyl as defined herein.

“Heteroalkyl” means an alkyl radical as defined herein wherein one, twoor three hydrogen atoms have been replaced with a substituentindependently selected from the group consisting of —OR^(a),—NR^(b)R^(c), and —S(O)_(n)R^(d) (where n is an integer from 0 to 2),with the understanding that the point of attachment of the heteroalkylradical is through a carbon atom, wherein R^(a) is hydrogen, acyl,alkyl, cycloalkyl, or cycloalkylalkyl; R^(b) and R^(c) are independentlyof each other hydrogen, acyl, alkyl, cycloalkyl, or cycloalkylalkyl; andwhen n is O, R^(d) is hydrogen, alkyl, cycloalkyl, or cycloalkylalkyl,and when n is 1 or 2, R^(d) is alkyl, cycloalkyl, cycloalkylalkyl,amino, acylamino, monoalkylamino, or dialkylamino. Representativeexamples include, but are not limited to, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxypropyl,1-hydroxymethylethyl, 3-hydroxybutyl, 2,3-dihydroxybutyl,2-hydroxy-1-methylpropyl, 2-aminoethyl, 3-aminopropyl,2-methylsulfonylethyl, aminosulfonylmethyl, aminosulfonylethyl,aminosulfonylpropyl, methylaminosulfonylmethyl,methylaminosulfonylethyl, methylaminosulfonylpropyl, and the like.

“Heteroaryl” means a monocyclic or bicyclic radical of 5 to 12 ringatoms having at least one aromatic ring containing one, two, or threering heteroatoms selected from N, O, or S, the remaining ring atomsbeing C, with the understanding that the attachment point of theheteroaryl radical will be on an aromatic ring. The heteroaryl ring maybe optionally substituted as defined herein. Examples of heteroarylmoieties include, but are not limited to, optionally substitutedimidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl,thiadiazolyl, pyrazinyl, thienyl, benzothienyl, thiophenyl, furanyl,pyranyl, pyridyl, pyrrolyl, pyrazolyl, pyrimidyl, quinolinyl,isoquinolinyl, benzofuryl, benzothiophenyl, benzothiopyranyl,benzimidazolyl, benzooxazolyl, benzooxadiazolyl, benzothiazolyl,benzothiadiazolyl, benzopyranyl, indolyl, isoindolyl, triazolyl,triazinyl, quinoxalinyl, purinyl, quinazolinyl, quinolizinyl,naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyland the like, including partially hydrogenated derivatives thereof, eachoptionally substituted.

“Heteroarylalkyl” or “heteroaralkyl” means a group of the formula —R—R′wherein R is alkylene and R′ is heteroaryl as defined herein.

“Heteroarylsulfonyl means a group of the formula —SO₂—R wherein R isheteroaryl as defined herein.

“Heteroaryloxy” means a group of the formula —O—R wherein R isheteroaryl as defined herein.

“Heteroaralkyloxy” means a group of the formula —O—R—R″ wherein R isalkylene and R′ is heteroaryl as defined herein.

The terms “halo”, “halogen” and “halide”, which may be usedinterchangeably, refer to a substituent fluoro, chloro, bromo, or iodo.

“Haloalkyl” means alkyl as defined herein in which one or more hydrogenhas been replaced with same or different halogen. Exemplary haloalkylsinclude —CH₂Cl, —CH₂CF₃, —CH₂CCl₃, perfluoroalkyl (e.g., —CF₃), and thelike.

“Haloalkoxy” means a moiety of the formula —OR, wherein R is a haloalkylmoiety as defined herein. An exemplary haloalkoxy is difluoromethoxy.

“Heterocycloamino” means a saturated ring wherein at least one ring atomis N, NH or N-alkyl and the remaining ring atoms form an alkylene group.

“Heterocyclyl” means a monovalent saturated moiety, consisting of one tothree rings, incorporating one, two, or three or four heteroatoms(chosen from nitrogen, oxygen or sulfur). The heterocyclyl ring may beoptionally substituted as defined herein. Examples of heterocyclylmoieties include, but are not limited to, optionally substitutedpiperidinyl, piperazinyl, homopiperazinyl, azepinyl, pyrrolidinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, pyridinyl, pyridazinyl,pyrimidinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinuclidinyl, quinolinyl, isoquinolinyl,benzimidazolyl, thiadiazolylidinyl, benzothiazolidinyl,benzoazolylidinyl, dihydrofuryl, tetrahydrofuryl, dihydropyranyl,tetrahydropyranyl, thiamorpholinyl, thiamorpholinylsulfoxide,thiamorpholinylsulfone, dihydroquinolinyl, dihydrisoquinolinyl,tetrahydroquinolinyl, tetrahydrisoquinolinyl, and the like.

“Heterocyclylalkyl” means a moiety of the formula —R—R′ wherein R isalkylene and R′ is heterocyclyl as defined herein.

“Heterocyclyloxy” means a moiety of the formula —OR wherein R isheterocyclyl as defined herein.

“Heterocyclylalkoxy” means a moiety of the formula —OR—R′ wherein R isalkylene and R′ is heterocyclyl as defined herein.

“Hydroxyalkoxy” means a moiety of the formula —OR wherein R ishydroxyalkyl as defined herein.

“Hydroxyalkylamino” means a moiety of the formula —NR—R′ wherein R ishydrogen or alkyl and R′ is hydroxyalkyl as defined herein.

“Hydroxyalkylaminoalkyl” means a moiety of the formula —R—NR′—R″ whereinR is alkylene, R′ is hydrogen or alkyl, and R″ is hydroxyalkyl asdefined herein.

“Hydroxycarbonylalkyl” or “carboxyalkyl” means a group of the formula—R—(CO)—OH where R is alkylene as defined herein.

“Hydroxyalkyloxycarbonylalkyl” or “hydroxyalkoxycarbonylalkyl” means agroup of the formula —R—C(O)—O—R—OH wherein each R is alkylene and maybe the same or different.

“Hydroxyalkyl” means an alkyl moiety as defined herein, substituted withone or more, preferably one, two or three hydroxy groups, provided thatthe same carbon atom does not carry more than one hydroxy group.Representative examples include, but are not limited to, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 2-hydroxy-1-hydroxymethylethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxypropyl

“Hydroxycycloalkyl” means a cycloalkyl moiety as defined herein whereinone, two or three hydrogen atoms in the cycloalkyl radical have beenreplaced with a hydroxy substituent. Representative examples include,but are not limited to, 2-, 3-, or 4-hydroxycyclohexyl, and the like.

“Urea” or means a group of the formula —NR′—C(O)—NR′R′″ wherein R′, R″and R′″ each independently is hydrogen or alkyl.

“Carbamate” means a group of the formula —O—C(O)—NR′R″ wherein R′ and R″each independently is hydrogen or alkyl.

“Carboxy” means a group of the formula —O—C(O)—OH.

“Sulfonamido” means a group of the formula —SO₂—NR′R″ wherein R′, R″ andR′″ each independently is hydrogen or alkyl.

“Optionally substituted”, when used in association with “aryl”, phenyl”,“heteroaryl” “cycloalkyl” or “heterocyclyl”, means an aryl, phenyl,heteroaryl, cycloalkyl or heterocyclyl which is optionally substitutedindependently with one to four substituents, preferably one or twosubstituents selected from alkyl, cycloalkyl, cycloalkylalkyl,heteroalkyl, hydroxyalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino,acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy,heteroalkyl, —COR, —SO₂R (where R is hydrogen, alkyl, phenyl orphenylalkyl), —(CR′R″)_(n)—COOR (where n is an integer from 0 to 5, R′and R″ are independently hydrogen or alkyl, and R is hydrogen, alkyl,cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or—(CR′R″)_(n)—CONR^(a)R^(b) (where n is an integer from 0 to 5, R′ and R″are independently hydrogen or alkyl, and R^(a) and R^(b) are,independently of each other, hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, phenyl or phenylalkyl).

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not.

“Inert organic solvent” or “inert solvent” means the solvent is inertunder the conditions of the reaction being described in conjunctiontherewith, including for example, benzene, toluene, acetonitrile,tetrahydrofuran, N,N-dimethylformamide, chloroform, methylene chlorideor dichloromethane, dichloroethane, diethyl ether, ethyl acetate,acetone, methyl ethyl ketone, methanol, ethanol, propanol, isopropanol,tert-butanol, dioxane, pyridine, and the like. Unless specified to thecontrary, the solvents used in the reactions of the present inventionare inert solvents.

“Solvates” means solvent additions forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The terms “those defined above” and “those defined herein” whenreferring to a variable incorporates by reference the broad definitionof the variable as well as preferred, more preferred and most preferreddefinitions, if any.

The term “emitting reporter” means a chemical substituent or materialthat produces, under appropriate excitation conditions, a detectableoptical signal. The optical signal produced by an emitting reporter istypically electromagnetic radiation in the near-infrared, visible, orultraviolet portions of the spectrum. The emitting reporters of theinvention are generally up-converting reporters, but can also be forexample, fluorescent and colorimetric substituents.

The term “phosphor particle” means a particle or composition comprisingat least one type of upconverting phosphor material.

The term “primer” means a nucleotide with a specific nucleotide sequencewhich is sufficiently complimentary to a particular sequence of a targetDNA molecule, such that the primer specifically hybridizes to the targetDNA molecule.

The term “probe” refers to a binding component which bindspreferentially to one or more targets (e.g., antigenic epitopes,polynucleotide sequences, macromolecular receptors) with an affinitysufficient to permit discrimination of labeled probe bound to targetfrom nonspecifically bound labeled probe (i.e., background).

The term “probe polynucleotide” means a polynucleotide that specificallyhybridizes to a predetermined target polynucleotide.

The term “oligomer” refers to a chemical entity that contains aplurality of monomers. As used herein, the terms “oligomer” and“polymer” are used interchangeably. Examples of oligomers and polymersinclude polydeoxyribonucleotides (DNA), polyribonucleotides (RNA), otherpolynucleotides which are C-glycosides of a purine or pyrimidine base,polypeptides (proteins), polysaccharides (starches, or polysugars), andother chemical entities that contain repeating units of like chemicalstructure.

The term “PCR” refers to polymerase chain reaction. This refers to anytechnology where a nucleotide is amplified via a temperature cyclingtechniques in the presence of a nucleotide polymerase, preferably a DNApolymerase. This includes but is not limited to real-time pertechnology, reverse transcriptase-per, and standard PCR methods.

The term “nucleic acid” means a polymer composed of nucleotides, e.g.deoxyribonucleotides or ribonucleotides, or compounds producedsynthetically which can hybridize with naturally occurring nucleic acidsin a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in hybridizationreactions, i.e., cooperative interactions through Pi electrons stackingand hydrogen bonds, such as Watson-Crick base pairing interactions,Wobble interactions, etc.

The terms “ribonucleic acid” and “RNA” as used herein mean a polymercomposed of ribonucleotides.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “polynucleotide” or “nucleotide” refer to single or doublestranded polymer composed of nucleotide monomers of generally greaterthan 50 nucleotides in length.

The term “monomer” as used herein refers to a chemical entity that canbe covalently linked to one or more other such entities to form anoligomer. Examples of “monomers” include nucleotides, amino acids,saccharides, peptides, and the like. The term nucleotide means

The term “linker” means a compound or a composition which covalentlylinks a biomolecule to the surface of a coated emitting reporter. Forexample, but not limited to a silyinated coated upconverting phosphorparticle linked to a DNA molecule.

The term “identifiable sequence” or “detectable sequence” means anucleotide sequence which can by detected by hybridization and/or PCRtechnology by a primer or probe designed for specific interaction withthe target nucleotide sequence to be identified. The interaction of thetarget nucleotide sequence with the specific probe or primer can bedetected by optical and/or visual means to determine the presence of thetarget nucleotide sequence.

Nomenclature and Structures

In general, the nomenclature used in this application is based onAUTONOM™ v.4.0, a Beilstein Institute computerized system for thegeneration of IUPAC systematic nomenclature. Chemical structures shownherein were prepared using ISIS® version 2.5. Any open valency appearingon a carbon, oxygen sulfur or nitrogen atom in the structures hereinindicates the presence of a hydrogen atom unless indicated otherwise.Where a chiral center exists in a structure but no specificstereochemistry is shown for the chiral center, both enantiomersassociated with the chiral center are encompassed by the structure.Where a structure shown herein may exist in multiple tautomeric forms,all such tautomers are encompassed by the structure.

Compounds of the Invention

The invention provides compounds of the formula I:(cOpR)-[L-(NA)]_(m)wherein:

m is an integer greater than 1;

(cOpR) is a coated optical reporter particle;

(NA) is a nucleic acid oligomer of detectable sequence; and

L is a linking group covalently bound to the coated optical reporterparticle and to the nucleic acid oligomer.

While formula I specifically relates to linking nucleic acid oligomersor nucleotides to the surface of the coated optical reporter particle,it should be understood to the those skilled in the art that otherbiomolecules besides nucleotides can be linked to L. Such biomoleculesinclude but are not limited to peptides, proteins, antibodies, enzymes,DNA binding proteins and the like. These biomolecules, may be modifiedto include lipids, carbohydrates, fluorescent and/or upconvertingphosphor molecules or other detectable compounds or markers.

In most embodiments, NA is a DNA oligomer. The DNA oligomer may beeither single stranded DNA or double stranded DNA. In certainembodiments NA may be comprise cDNA, RNA, STR (single tandem repeat) orSNP (single nucleotide polymorphism). NA oligomers of the compositionsof the invention may also be modified to comprise at least one dUTPnucleic acid or at least one nucleic acid within the oligomer which hasbeen modified to contain a detectable marker.

In many embodiments NA is a DNA oligomer having a length of betweenabout 40 base pairs and about 1000 base pairs (per strand).

In other embodiments the DNA has a length of between about 80 and 500base pairs (per strand).

In yet other embodiments the DNA has a length of between about 100 toabout 250 base pairs (per strand).

The DNA used with the invention may be natural or syntheticallyproduced. All or a portion of the DNA may comprise an identifiablesequence.

In certain embodiments of formula I, the coated optical reportercomprises a visible or infrared detectable light emitting materialselected from the group consisting of a fluorescent dye, an upconvertingphosphor, a ceramic powder, or a quantum dot material. In mostembodiments where the cOpR comprises a visible or infrared detectablelight emitting material, the light emitting materials are excitable byUV, visible or an infrared light source.

In some embodiments, rare earth-doped ceramic particles are used asphosphor particles. Phosphor particles may be detected by any suitablemethod, including but not limited to up-converting phosphor technology(UPT), in which up-converting phosphors transfer lower energy infrared(IR) radiation into higher-energy visible light. Although anunderstanding of the mechanism is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action, in some embodiments the UPT up-converts infraredlight to visible light by multi-photon absorption and subsequentemission of dopant-dependant phosphorescence (See, e.g., U.S. Pat. No.6,399,397; van De Rijke, et al., Nature Biotechnol. 19(3):273-6 (2001);Corstjens, et al., IEE Proc. Nanobiotechnol. 152(2):64 (2005), eachincorporated by reference herein in its entirety.

In many embodiments, the phosphor nanoparticles may be of the formula A(Y_(x)RE¹ _(y),RE² _(z))₂O₃  Awherein:

RE¹ and RE² each is a different rare earth element;

x is from about 0.6 to about 0.95;

y is from 0 to about 0.35; and

z is from 0 0.1 to about 0.001;

provided that y and z are not simultaneously equal to 0.

The rare earth elements RE¹ and RE² may each independently be selectedfrom Ytterbium, Erbium, Holmium, Thulium, or Terbium.

In many embodiments RE¹ is Ytterbium.

In many embodiments RE² is Erbium.

The up-converting particles utilized in the methods of the invention maybe spherical, non-agglomerated, non-porous particles with an averagesize of 40-60 nm. In general, particle sizes may range from about 10 nmto about 5 um in size. Such up-converting phosphor nanopowders such asdoped yttrium oxide and yttrium oxysulfide are commercially availableand may be obtained from such as Nanocerox, Inc., of Ann Arbor, Mich.

Suitable examples of up-converting phosphors are compounds of rareearths or elements from the group IIIB such as: Na-yttrium fluoride,lanthanum fluoride, lanthanum oxysulphide, yttrium oxysulphide, yttriumfluoride, yttrium gallate, gadolinium fluoride, barium-yttriumfluorides, gadolinium oxysulphide as well as compounds of the above typedoped with activator pairs such as ytterbium/erbium, ytterbium/thuliumor ytterbium/holmium. Other suitable up-converting phosphors includechelate compounds of erbium, neodymium, thulium, holmium andpraseodymium.

The following compositions are merely illustrative of some of theup-converting phosphor containing compositions that can be synthesizedby the synthetic reaction schemes of the methods of the presentinvention. Various modifications to these synthetic reaction schemes canbe made and will be suggested to one skilled in the art having referredto the disclosure contained in this application.

TABLE I Upconverting Phosphor Compositions Phosphor Material AbsorberIon Emitter Ion Oxysulfides (O₂S) Y₂O₂S Ytterbium Erbium Gd₂O₂SYtterbium Erbium La₂O₂S Ytterbium Holmium Oxyhalides (OX_(y)) YOFYtterbium Thulium Y₃OCl₇ Ytterbium Terbium Fluorides (F_(x)) YF₃Ytterbium GdF₃ Ytterbium Erbium LaF₃ Ytterbium Erbium NaYF₃ YtterbiumHolmium BaYF₅ Ytterbium Thulium BaY₂F₈ Ytterbium Thulium Gallates(Ga_(x)O_(y)) YGaO₃ Ytterbium Erbium Y₃Ga₅O₁₂ Ytterbium Erbium Silicates(Si_(x)O_(y)) YSi₂O₅ Ytterbium Holmium YSi₃O₇ Ytterbium Thulium

In certain embodiments the coated optical reporter may also comprise atleast one electromagnetic emitting material. An electromagnetic emittingmaterial as part of the composition of the invention, allows for thecomposition to be detected by various methods and devices. Where theelectromagnetic emitting material is detectable by mechanical deviceswhich provide at least one source selected from the group consisting ofan infrared radiation source, magnetic field source or electromagneticpulse. This electromagnetic emitting material may be in conjunction withat least one light emitting material, such as an upconverting phosphor.

When the compositions of the invention comprise UCPs, the upconvertingphosphor material/particle in certain embodiments have the formula BY_(x)Yb_(y)Er_(z)O₂S  Bwherein:

x is from about 0.6 to about 0.95;

y is from about 0.05 to about 0.35; and

z is from about 0.1 to about 0.001.

In other embodiments, the upconverting phosphor particle may be of theformula C:Na(Y_(x)Yb_(y)Er_(z))F₄  Cwherein

x is from about 0.6 to about 0.95

y is from about 0.05 to about 0.35; and

z is from about 0.1 to about 0.001.

In certain embodiments of formula I, L comprises an alkylene moietyhaving a first end covalently bound to the coated optical reporterparticle (cOpR) and a second end covalently bound to the nucleic acidoligomer (NA).

In many embodiments of formula I, L is of the formula D:-A-R¹—B—  Dwherein:

R¹ is C₂₋₈alkylene;

-A- is a group covalently bonded to the surface of the coated opticalreporter; and

—B— is a group covalently bonded to the 3′ or 5′ end of the nucleic acidoligomer.

In certain embodiments of formula D, —R¹— is —(CH₂)_(n)— and n is from 2to 8.

In certain embodiments of formula D, —B— is:—S—;—O—;—NR^(a)—;—S—(CH₂)_(p)—;—O—(CH₂)_(p)—;—NR^(a)—(CH₂)_(p)—;—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—O—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—NR^(a)—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—S—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;—O—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—; or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;

wherein:

-   -   p is from 2 to 8;    -   q is from 1 to 8;    -   r is from 2 to 8; and    -   each R^(a) is independently hydrogen or a C₁₋₆alkyl.

In certain embodiments of formula D, —B— is:—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p) or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;

wherein:

-   -   p is from 2 to 8;    -   q is from 1 to 8;    -   r is from 2 to 8; and    -   each R^(a) is independently hydrogen or a C₁₋₆alkyl.

In other embodiments of formula D, —B— is:—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p) or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR²—(CH₂)_(p)—;

wherein:

-   -   p is from 2 to 6;    -   q is from 1 to 3; and    -   r is 2 or 3.

In other embodiments of formula D, —B— is—S—CH₂—C(O)—NH—(CH₂)₆—or—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆—.

In certain embodiments of formula D, -A- is —O—.

In many embodiments of formula I, the coated optical reporter (cOpR) iscoated with silica. Usually when the coated optical reporter comprises acoating of silica, the silica comprises at least one Si—O bond.

The value of m in formula I will vary according to the surface area ofthe coated optical reporter and the number of functional groups on theoptical reporter surface cable of bonding to -L-. The value of m isalways greater than one, and usually greater than 10. Preferably m isgreater than 100, and in many embodiments m is greater than 10³. In manyembodiments m may be, for example, between about 10 and about 10⁹. Incertain embodiments m may be from about 100 to about 10⁸. In someembodiments m may be from about 10³ to about 10⁷.

In certain embodiments the compositions of the invention are of theformula II:(UCP)-[A-R¹—X—R²—C(O)—NR^(a)—R³-(DNA)]_(m)  IIwherein:

m is an integer greater than 1;

UCP is an upconverting phosphor particle;

DNA is a single or double stranded deoxyribonucleic acid oligomer;

-A- is a group capable of covalently bonding to the surface of the

Upconverting phosphor particle;

R¹ is C₂₋₈alkylene,

R² is C₁₋₈alkylene or —C(O)—C₁₋₈alkylene-;

—X— is —O—, —S— or —NR^(a)—;

R³ is C₂₋₈alkylene; and

R^(a) is hydrogen or C₁₋₆alkyl.

In certain embodiments of the invention, the subject composition may beof formula III:(UCP)—[O—R¹—X—R²—C(O)—NH—R³-DNA]_(m)  IIIwherein m, R¹, R², R³, UCP and DNA are as defined herein.

In certain embodiments of the invention, R¹ is C₂₋₆alkylene.

In certain embodiments of the invention, R² is C₁₋₆alkylene.

In certain embodiments of the invention, R³ is C₂₋₆alkylene.

In certain embodiments of the invention, R² is —C(O)—C₂₋₆alkylene-.

In certain embodiments of the invention, R¹ is —(CH₂)_(s)— wherein s isfrom 2 to 6. In some embodiments s is 3.

In certain embodiments of the invention, R² is —(CH₂)_(t)— wherein t isfrom 1 to 6. In some embodiments t is 1.

In certain embodiments of the invention, R² is —C(O)—(CH₂)_(u)— whereinu is from 1 to 6. In some embodiments u is 2 or 3, preferably 2.

In certain embodiments of the invention, R³ is —(CH₂)_(v)— wherein v isfrom 2 to 6. In some embodiments v is 6.

In certain embodiments of the invention, the subject composition may beof formula IV:(UCP)—[O—(CH₂)_(s)—S—(CH₂)_(t)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  IV

wherein:

-   -   s is from 2 to 6;    -   v is from 2 to 6;    -   t is from 1 to 3; and    -   m, UCP and DNA are as defined herein.

In certain embodiments of the invention, the compositions may be offormula V:(UCP)—[O—(CH₂)_(s)—NH—C(O)—(CH₂)_(u)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  V

wherein:

-   -   s is from 2 to 6;    -   v is from 2 to 6;    -   u is 2 or 3; and    -   m, UCP and DNA are as defined herein.

In certain embodiments of the invention, the compositions may be offormula VI:(UCP)—[O—(CH₂)₃—S—CH₂—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VI

wherein m, UCP and DNA are as defined herein.

In certain embodiments of the invention, the compositions may be offormula VII:(UCP)—[O—(CH₂)₃—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VII

wherein m, UCP and DNA are as defined herein.

Synthesis

Nucleotide-labeled optical reporters in accordance with the inventioncan be made by a variety of methods, including those depicted in theillustrative synthetic reaction schemes shown and described below.

Reagents and solvents used in preparing these compounds generally areeither available from commercial suppliers, such as Aldrich ChemicalCo., or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis; Wiley & Sons: New York, 1991, Volumes1-15; Rodd's Chemistry of Carbon Compounds, Elsevier Science Publishers,1989, Volumes 1-5 and Supplementals; and Organic Reactions, Wiley &Sons: New York, 1991, Volumes 1-40.

The starting materials and some of the intermediates of the syntheticreaction schemes can be isolated and purified if desired usingconventional techniques, including but not limited to, filtration,distillation, crystallization, chromatography, and the like.

Unless specified to the contrary, the reactions described hereinpreferably are conducted under an inert atmosphere at atmosphericpressure at a reaction temperature range of from about −78° C. to about150° C., more preferably from about 0° C. to about 125° C., and mostpreferably and conveniently at about room (or ambient) temperature,e.g., about 20° C.

Scheme A below illustrates one synthetic procedure usable to preparecertain compositions of the invention, wherein R is lower alkyl and maybe the same or different in each occurrence, R^(d) is hydrogen or loweralkyl, Y is a leaving group, Ag is an activating group, and m, X, R^(a),R¹, R² and R³ are as defined herein

In step 1 of Scheme A, UCP particles a are treated or reacted with amixture of alkoxysilicate reagents b and c, to form a functionalized UCPparticles d. In many embodiments R may be ethyl, such that reagent a istetraethyl orthosilicate. The ratio of reagents a and b may be varied tovary the value m. In certain embodiments R¹ may be —(CH₂)₃— and —XH maybe —SH or —NR^(a)H wherein R^(a) is hydrogen or lower alky.

In step 2, functionalized particles d are reacted with compound e toafford carboxylate ester-functionalized UCPs f. Y may be halo, mesyl,tosyl, or other suitable leaving groups. In certain embodiments whereinR² is —C(O)—C₁₋₈alkylene-, reagent e may be replaced with a cyclicanhydride such as succinic anhydride, glutaric anhydride, oxetane2,3-dione, or like anhydride.

The functionalized UCPs f are activated in step 4 by treating UCPs fwith an activating agent AA to form functionalized activated UCPs g. Theactivating agent in many embodiments may be N-hydroxy-succinimide orN-bromo-succinimide such that the group OR in particles f is replaced bya succimido activating group. Other possible activating groups includebut are not limited to Nitrophenyl ester.

In step 5 the activated particles are reacted with DNA reagent h to formDNA-labeled UCPs i. The DNA reagent h may comprise, for example, asingle stranded DNA oligomer of known sequence having an aminoalkylgroup at the 3′ or 5′ end that is capable of amide coupling with theactivated carboxy functional groups on particles h. Various amidecoupling techniques may thus be used in step 4, includingcarbodiimide-based amide couplings. The i of step 4 are a composition offormula I in accordance with the invention.

Many variations on the procedure of Scheme A are possible and willsuggest themselves to those skilled in the art. Specific details forproducing compositions of the invention are described in the Examplessection below.

Utility

The methods of the invention are useful in producing compoundscomprising biomolecules such as dsDNA, ssDNA, STR, SNP's as well apeptides and proteins.

The compounds of the invention are usable as authentication markers forvarious articles. For example, the compounds of the invention can beplaced in or on such articles as clothing, paintings, documents,medicines, industrial solutions, computer components, explosives and thelike. The compounds produced can also be utilized in micro arraytechnology, as well as protein expression, genomic identification andother technologies utilizing DNA hybridization techniques.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Unless otherwise stated, all temperatures including melting points(i.e., MP) are in degrees Celsius (° C.). It should be appreciated thatthe reaction which produces the indicated and/or the desired product maynot necessarily result directly from the combination of two reagentswhich were initially added, i.e., there may be one or more intermediateswhich are produced in the mixture which ultimately leads to theformation of the indicated and/or the desired product. The followingabbreviations may be used in the Preparations and Examples.

Up-converting phosphor nanopowder (doped yttrium oxide and yttriumoxysulfide upconverting particles) were obtained from Nanocerox, Inc.,Ann Arbor, Mich.

ABBREVIATIONS

-   -   UCP Up converting phosphor    -   UTP Up converting phosphor technology    -   OpR optical reporter particle    -   cOpR coated optical reporter particle    -   TEOS tetraethoxysilane, tetraethyl orthosilicate; ethyl        silicate; silicic acid, tetraethyl ester; silicon ethoxide    -   MOS methyl oxysilane    -   EOS ethyl oxysilane    -   POS propyl oxysiline    -   NHS N-Hydroxsuccinimde    -   IOA Iodoacetamide    -   DIPCI Diisopropylcarbodiimide    -   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCM dichloromethane/methylene chloride    -   DIPEA diisopropyl ethylamine    -   DME 1,2-dimethoxyethane (glyme)    -   DMF N,N-dimethylformamide    -   DMAP 4-dimethylaminopyridine    -   ECDI 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide    -   EtOAc ethyl acetate    -   EtOH ethanol    -   GC Gas Chromatography    -   HMPA hexamethylphosphoramide    -   HOBt N-Hydroxybenzotriazole    -   hplc high performance liquid chromatography    -   mCPBA m-chloroperbenzoic acid    -   MeCN acetonitrile    -   NMP N-methylpyrrolidinone    -   TEA triethylamine    -   THF tetrahydrofuran    -   LDA lithium diisopropylamine    -   TLC thin layer chromatography

Example 1 Doped Yttrium Oxysulfide withOxypropylsulfanylacetamide-Linked DNA

The synthetic procedure of this Example is shown below in Scheme B.

The method utilized in this example generally involved initially coatingthe upconverting phosphor particles with tetraethyl orthosilicate withammonium hydroxide present as a catalyst followed by hydrolysis withthiotriethoxysilane.¹ The coated particles were then treated withiodoacetic acid under basic conditions. The particles now modified withcarboxylic acid groups were activated using N-hydroxysuccinimide (NHS)and diisopropylcarbodiimide (DIPCI). Subsequently after activation,amino C₆ DNA was added to the mixture, for DNA attachment to thephosphor coated particles. The particles were isolated and washedextensively with copious amounts of water removing unreacted amino C₆DNA and other reagents. The isolated phosphor particles were thenevaluated by PCR to determine the detectable presence of the covalentlyattached DNA bound to the coated nanophosphor particles.

General

Doped yttrium oxide and yttrium oxysulfide upconverting particles werefrom Nanocerex (Ann Arbor, Mich.). Tetraethoxysilane was 99+% and3-aminopropyltriethoxysilane was 99% both from Aldrich.3-Mercaptopropyltriethoxysilane was 95% from Gelest and was lot#3C-2709. N-Hydroxsuccinimde, glutaric anhydride and iodoacetic acidwere from Aldrich. The 5′ C6 amino modified DNA was a 180-mer with amolecular weight of 55,867 and was obtained from Integrated DNATechnologies. In general, the 5′ amino modified DNA molecule is producedby modifying the 5′ end of the last sugar molecule of the DNA chain viaan OH and ultimately forms an ether linkage forming[sugar]-O—(CH₂)₆—NH₂. The concentration of the modified DNA was 1 nM.The 5′ amino linker portion of the modified DNA can range from twocarbons to about 18 carbons in length. 2-Propanol was HPLC grade fromAldrich or Fisher. The ultrasonic cleaning bath was a HeatSystems-Ultrasonics.

Particles were isolated by centrifuging using screw capped 50 mLpolypropylene Falcon tubes and a ICE Model HN—S benchtop centrifuge.

Step 1 Mercaptopropyl Functionalized Silylated Yttrium OxysulfideParticles:

Yttrium oxysulfide upconverting particles (100 mg) were suspended in 100mL of de-aerated 2-propanol and sonicated under nitrogen for 75 min.De-aerated water (7.5 mL) was added and sonication continued for 5 min.Concentrated ammonium hydroxide (9 mL) was added and sonicationcontinued a final 10 min. The milky white suspension was placed in anoil bath at 40±2° and stirred magnetically under nitrogen for 10 min.Tetraethoxysilane (0.3 mL) was added the mixture stirred for 2 hr.3-Mercaptopropyltriethoxysilane (0.3 mL) was added and stirringcontinued for 2.5 hr. The particles were isolated by centrifuging andwashed with 30 mL of 2-propanol. They were then washed with 20 mL of 5%acetic acid in 2-propanol and finally with 20 mL of 2-propanol. Theparticles were dried under vacuum (1.0 mm) at room temperatureovernight.

Step 2 Reaction of Thiol Functionalized Yttrium Oxysulfide Particleswith Iodoacetic Acid:

Iodoacetic acid (50 mg, 0.27 mmol) was dissolved in 20 mL of 0.2Maqueous sodium bicarbonate. The pH of the solution was about 8.4 (narrowrange pH paper). The thiol functionalized particles (˜100 mg) from step1 were added and the mixture sonicated in the dark under nitrogen for 10hr. An additional 50 mg of iodoacetic acid was added and sonicationcontinued overnight. During this time the bath temperature rose to 40°.The particles were isolated by centrifuging and washed with 10 mL ofwater. They were then sonicated with 10 mL of 10% aqueous acetic acid.The particles were isolated by centrifuging, washed with 10 mL of waterand vacuum dried overnight. The particles were examined by transmissionFTIR (KBr disk). There was a strong Si—O absorption at 1100 cm⁻¹.

Step 3 Activation of the Carboxy Yttrium Oxysulfide Particles withN-Hydroxysuccinimide:

The carboxy yttrium oxysulfide particles from step 2 (˜100 mg) weresuspended in 10 mL of 2-propanol and sonicated for 90 min.N-Hydroxysuccinimide (58 mg, 0.5 mmol) was added and sonicationcontinued for 5 min. The suspension was transferred to a 20 mLscintillation vial and diisopropylcarbodiimide (80 uL, 0.5 mmol) addedwith stirring. The mixture was stirred at room temperature (25°)overnight (15 hr). The particles were isolated by centrifuging thenwashed by centrifuging with 10 mL of 2-propanol and then twice with 5 mLof the same solvent. They were finally dried in vacuum at roomtemperature for about 5 min and used immediately in the next step forreaction with DNA.

Step 4 Treatment of the Activated Particles with Amino Modified DNA:

The activated yttrium oxysulfide particles from step 3 (˜50 mg) weresuspended in 5 mL of 50 mM borate buffer (pH 8.45) using a Vortex mixer.The amino functionalized long chain DNA (2 nmole) in 50 uL of water wasadded and the mixture was stirred magnetically at room temperature for24 hr. The particles were isolated by centrifuging and washed with 5 mLof the borate buffer and then with twice with 5 mL of water. They weresuspended in 2 mL of water and stored at 5°.

Example 1 Alternate Procedure

Step 1 Mercaptopropyl Functionalized Silylated Yttrium Oxide Particles:

Yttrium oxide upconverting particles (100 mg) were suspended in 100 mLof de-aerated 2-propanol and sonicated under nitrogen for 90 min.De-aerated water (7.5 mL) was added and sonication continued for 5 min.Concentrated ammonium hydroxide (9 mL) was added and sonicationcontinued a final 10 min. The milky white suspension was placed in anoil bath at 40±2° and stirred magnetically under nitrogen for 10 min.Tetraethoxysilane (0.3 mL) was added and the mixture stirred for 2 hr.3-Mercaptopropyltriethoxysilane (0.3 mL) was added and stirringcontinued for 2.5 hr. The particles were isolated by centrifuging andwashed with 40 mL of 2-propanol. They were then washed with 20 mL of 5%acetic acid in 2-propanol and finally with 20 mL of 2-propanol. Theparticles were dried under vacuum (1.0 mm) at room temperature (20°overnight.

Step 2 Reaction of Thiol Functionalized Yttrium Oxide Particles withIodoacetic Acid:

The thiol functionalized yttrium oxide particles from step 2 (˜90 mg)were suspended in 20 mL of 0.2M de-aerated aqueous sodium bicarbonateand the mixture sonicated in the dark under nitrogen for 15 min. Theparticles were transferred to a flask and iodoacetic acid (50 mg, 0.27mmol) added. The suspension was stirred magnetically at 40±2° undernitrogen for 5 hr. An additional 50 mg of iodoacetic acid was added andstirring continued overnight (14 hr). The particles were isolated bycentrifuging and washed with 10 mL of water. They were then sonicatedwith 10 mL of 10% aqueous acetic acid for 10 min. The particles wereisolated by centrifuging, washed with 10 mL of water and vacuum driedovernight. The particles were examined by transmission FTIR (KBr disk).There was a strong Si—O absorption at 1100 cm⁻¹.

Step 3 Activation of the Carboxy Yttrium Oxide Particles withN-Hydroxysuccinimide:

The carboxy yttrium oxide particles of step 2 (˜80 mg) were suspended in10 mL of 2-propanol and sonicated for 60 min. N-Hydroxysuccinimide (58mg, 0.5 mmol) was added and sonication continued for 10 min. Thesuspension was transferred to a 20 mL scintillation vial anddiisopropylcarbodiimide (80 uL, 0.5 mmol) added with stirring. Themixture was stirred at room temperature (˜25°) overnight (18 hr). Theparticles were isolated by centrifuging then washed by centrifuging with10 mL of 2-propanol and then with 2×5 mL of the same solvent. They werefinally dried in vacuum at room temperature for about 5 min and usedimmediately in the next step for reaction with DNA.

Step 4 Treatment of the Activated Yttrium Oxide Particles with AminoModified DNA:

The activated particles from step 3 (˜80 mg) were suspended in 10 mL of50 mM borate buffer (pH 8.45) using a Vortex mixer. The aminofunctionalized long chain DNA (1 nmole) in 50 uL of water was added andthe mixture and stirred magnetically at room temperature for 22 hr. TheDNA linker in this example was 6 carbons long, and the single strandedDNA oligomer comprised 180 bases. The particles were isolated bycentrifuging and washed with 10 mL of the borate buffer and then withtwice with 10 mL of water. They were suspended in 2 mL of water andstored at 5°.

Example 2 Doped Yttrium Oxysulfide with (oxy-propylamino)-acetic acid5-amino-4-oxo-pentyl ester-linked DNA

The synthetic procedure of this example is shown below in Scheme C.

The method utilized in this example generally involved initially coatingthe upconverting phosphor particles with tetraethyl orthosilicate withammonium hydroxide present as a catalyst followed by hydrolysis withthiotriethoxysilane. The coated particles were then treated withiodoacetic acid under basic conditions. The particles now modified withcarboxylic acid groups were activated using N-hydroxysuccinimide (NHS)and diisopropylcarbodiimide (DIPCI). Subsequently after activation,amino C₆ DNA was added to the mixture, for DNA attachment to thephosphor coated particles. The particles were isolated and washedextensively with copious amounts of water removing unreacted amino C₆DNA and other reagents. The isolated phosphor particles were thenevaluated by PCR to determine the detectable presence of the covalentlyattached DNA bound to the coated nanophosphor particles.

Step 1 Aminopropyl Functionalized Silylated Yttrium OxysulfideParticles:

Yttrium oxysulfide upconverting particles (100 mg) were suspended in 100mL of de-aerated 2-propanol and sonicated under nitrogen for 75 min.De-aerated water (7.5 mL) was added and sonication continued for 10 min.Concentrated ammonium hydroxide (9 mL) was added and sonicationcontinued a final 10 min. The milky white suspension was placed in anoil bath at 40±2° and stirred magnetically under nitrogen for 10 min.Tetraethoxysilane (0.3 mL) was added and the mixture stirred for 2 hr.3-Aminopropyltriethoxysilane (0.3 mL) was added and stirring continuedfor 2.7 hr. The particles were isolated by centrifuging and washed threetimes with 20 mL of 2-propanol. The particles were dried under vacuum(1.0 mm) at room temperature (30°) overnight. Analysis by transmissionFTIR (KBr disk) showed a strong Si—O absorption at 1100 cm⁻¹. FIG. 1 isan FTIR spectrum of yttrium oxysulfide upconverting particles aftertreatment of with TEOS for 2 hours, followed by 3-aminopropyltriethoxysilane for 12 hours. FIG. 1 shows the strong absorption of theSi—O bond at 1100 cm⁻¹, demonstrated the silination of the UCPparticles.

Step 2 Reaction of the Amino Functionalized Yttrium Oxysulfide Particleswith Glutaric Anhydride:

Glutaric anhydride (0.34 g, 3.0 mmol) was dissolved in 13 mL of2-propanol (minimum volume needed at room temperature) (30°). Thesolution was added to the amino functionalized yttrium oxysulfideparticles (90 mg) and the mixture sonicated for an hour at 30-35°. Thesuspension was transferred to a flask and stirred magnetically at 40°for 24 hr. The particles were isolated by centrifuging and washed threetimes with 10 mL of 2-propanol then dried under vacuum at roomtemperature (26°) overnight.

Step 3 Activation of the Carboxy Yttrium Oxysulfide Particles withN-Hydroxysuccinimide:

The previous carboxy yttrium oxysulfide particles (˜80 mg) weresuspended in 10 mL of 2-propanol and sonicated for 60 min.N-Hydroxysuccinimide (58 mg, 0.5 mmol) was added and sonicationcontinued for 5 min. The suspension was transferred to a 20 mLscintillation vial and diisopropylcarbodiimide (80 uL, 0.5 mmol) addedwith stirring. The mixture was stirred at room temperature (˜23°)overnight (21 hr). The particles were isolated by centrifuging thenwashed by centrifuging with 10 mL of 2-propanol and then twice with 5 mLof the same solvent. They were finally dried in vacuum at roomtemperature for about 5 min and used immediately in the next step forreaction with DNA.

Step 4 Treatment of the Activated Yttrium Oxysulfide Particles withAmino Modified DNA:

The activated particles (˜80 mg) were suspended in 10 mL of 50 mM boratebuffer (pH 8.45) using a Vortex mixer. The amino functionalized longchain DNA (1 nmole) in 50 uL of water was added and the mixturesonicated for a few minutes. The DNA linker in this example was 6carbons long, and the single stranded DNA oligomer comprised 180 bases.It was transferred to a 20 mL scintillation vial and stirredmagnetically at room temperature (22°) for 20 hr. The particles wereisolated by centrifuging and washed with 10 mL of the borate buffer andthen three times with 10 mL of water. They were suspended in 2.5 mL ofwater and stored at 5°.

Example 3

This example demonstrates that the compositions produced by the methodsof the inventions, particularly those methods in which nucleotides arelinked to a phosphor, that the nucleotide attached to the compositioncan be detected directly by techniques such as PCR. The phosphorcompound utilized in this example was Yttrium oxysulfide as well as anamine linked DNA oligomer.

Detection of Bound DNA to Phosphor Particles by Real-Time-PCR.

The equipment and supplies utilized for RT-PCR were the following. PCRcapillary system (20 ul capillary) by Roche Diagnostics, LightCycler 2by Roche Diagnostics, SYBR Green ReadyMix RT-PCR kit by Sigma-Aldrichand SYBR Green JumpStart Tag mix by Sigma.

The following primers were specifically designed for amplification ofthe DNA oligomer attached to the phosphor particles produced by themethods of the invention. Primer 1-(5′-CGCCAGGGT TTTCCCAGTCACGAC-3′) andPrimer 2 (5′-CAGGAAACAGCTATGAC-3′). The final concentration of theprimers for RT-PCR amplification was 0.05 uM in the RT-PCR r×n sample.The size of the amplicon generated during RT-PCR with this primer pairwas approximately 150 bp in length.

The RT-PCR run conditions were as follows. One pre-heating cycle of 95°C. for 5 minutes, followed by 40 cycles of 20 seconds at 95° C., 40seconds at the annealing temperature of 50° C., with polymeraseextension at 72° C. for 20 seconds.

The isolated phosphor particles containing DNA molecules wereresuspended in and diluted 1/10, 1/100, and 1/1000, respectively forRT-PCR analysis. Each RT-PCR sample contained 15 ul of RT-PCR master mix0.5 ul of each Primer stock solution, 1 ul of a specified dilutedphosphor containing DNA sample, and 13 ul water were mixed and put into20 ul capillary tubes. Positive and Negative controls were alsoprepared. Duplicates of all RT-PCR samples were prepared and analyzed.

The results from the RT-PCR experiment are shown in FIG. 2. The resultsin FIG. 2 show that the 1/100 dilution sample had a Ct of 30, while the1/10 and 1/1000 dilution had a Ct of 33 and Ct of 36, respectively. Atthe 1/10 dilution the concentration of the UCP particles is high enoughto quench the PCR signal, thus delaying the cycle in which amplificationof the target DNA is present.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A composition of the formula I:(cOpR)-[L-(NA)]_(m)  I wherein: m is an integer greater than 1; (cOpR)is a coated optical reporter particle, wherein the cOpR comprises anupconverting phosphor (UCP) material of the formulaNa(Y_(x)Yb_(y)Er_(z))F₄, and wherein: x is from about 0.6 to about 0.95;y is from about 0.05 to about 0.35; and z is from about 0.1 to about0.001; (NA) is a nucleic acid oligomer of detectable sequence; and L isa linking group covalently bound to the coated optical reporter particleand to the nucleic acid oligomer; provided that at least one of thefollowing applies: x is not from 0.7 to 0.9; and/or y is not from 0.08to 0.29; and/or z is not from 0.01 to 0.06.
 2. The composition of claim1, wherein (NA) is a single stranded DNA molecule having a length ofbetween about 40 nucleotides and about 1000 nucleotides or a doublestranded DNA molecule having a length of between about 40 base pairs andabout 1000 base pairs.
 3. The composition of claim 1, wherein Lcomprises an alkylene moiety having a first end covalently bound to thecoated optical reporter particle and a second end covalently bound tothe nucleic acid oligomer.
 4. The composition of claim 1, wherein L isof the formula:-A-R¹—B— wherein: R¹ is C₂₋₈alkylene; -A- is a group covalently bondedto the surface of the coated optical reporter; and —B— is a groupcovalently bonded to the 3′ or 5′ end of the nucleic acid oligomer. 5.The composition of claim 4, wherein -A- is —O—.
 6. The composition ofclaim 4, wherein —R¹— is —(CH₂)_(n)— and wherein n is from 2 to
 8. 7.The composition of claim 4, wherein —B— is:—S—;—O—;—NR^(a)—;—S—(CH₂)_(p)—;—O—(CH₂)_(p)—;—NR^(a)—(CH₂)_(p)—;—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—O—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—NR^(a)—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p)—;—S—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—;—O—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—; or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p)—; wherein: p is from 2 to8; q is from 1 to 8; r is from 2 to 8; and each R^(a) is independentlyhydrogen or C₁₋₆alkyl.
 8. The composition of claim 4, wherein —B— is:—S—(CH₂)_(q)—C(O)—NR^(a)—(CH₂)_(p) or—NR^(a)—C(O)—(CH₂)_(r)—C(O)—NR^(a)—(CH₂)_(p); wherein: p is from 2 to 8;q is from 1 to 8; r is from 2 to 8; and each R^(a) is independentlyhydrogen or C₁₋₆alkyl.
 9. The composition of claim 8, wherein: p is from2 to 6; q is from 1 to 3; and r is 2 or
 3. 10. The composition of claim4, wherein —B— is:—S—CH₂—C(O)—NH—(CH₂)₆—; or—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆—.
 11. The composition of claim 1, whereinthe cOpR is coated with silica.
 12. A composition of the formula II:(UCP)-[A-R¹—X—R²—C(O)—NR^(a)—R³-(DNA)]_(m)  II wherein: m is an integergreater than 1; UCP is an upconverting phosphor particle of the formulaNa(Y_(x)Yb_(y)Er_(z))F₄, wherein: x is from about 0.6 to about 0.95; yis from about 0.05 to about 0.35; and z is from about 0.1 to about0.001; DNA is a single or double stranded deoxyribonucleic acidoligomer; -A- is a group covalently bound to a surface of the UCP; R¹ isC₂₋₈alkylene, R² is C₁₋₈alkylene or —C(O)—C₁₋₈alkylene-; —X— is —O—, —S—or —NR^(a)—; R³ is C₂₋₈alkylene; and R^(a) is hydrogen or C₁₋₆alkyl;provided that at least one of the following applies: x is not from 0.7to 0.9; and/or y is not from 0.08 to 0.29; and/or z is not from 0.01 to0.06.
 13. The composition of claim 12, wherein the composition is of theformula IV:(UCP)—[O—(CH₂)_(s)—S—(CH₂)_(t)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  IV wherein:s is from 2 to 6; v is from 2 to 6; and t is from 1 to
 3. 14. Thecomposition of claim 12, wherein the composition is of the formula V:(UCP)—[O—(CH₂)_(s)—NH—C(O)—(CH₂)_(u)—C(O)—NH—(CH₂)_(v)-(DNA)]_(m)  Vwherein: s is from 2 to 6; v is from 2 to 6; and u is 2 or
 3. 15. Thecomposition of claim 12, wherein the composition is of the formula VI:(UCP)—[O—(CH₂)₃—S—CH₂—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VI.
 16. The compositionof claim 12, wherein the composition is of the formula VII:(UCP)—[O—(CH₂)₃—NH—C(O)—(CH₂)₃—C(O)—NH—(CH₂)₆-(DNA)]_(m)  VII.
 17. Thecomposition of claim 1, wherein the cOpR comprises a visually detectablelight emitting material selected from the group consisting of afluorescent dye, a ceramic powder, or a quantum dot.
 18. The compositionof claim 17, where said light emitting materials are excitable by a UVlight source or an infrared light source.
 19. The composition of claim1, wherein the cOpR comprises at least one electromagneticradiation-emitting material.
 20. The composition of claim 19, where theelectromagnetic radiation-emitting material is detectable by deviceswhich provide sources selected from the group consisting of an infraredradiation source, magnetic field source, a quantum dot orelectromagnetic pulse.
 21. A composition of the formula I:(cOpR)-[L-(NA)]_(m)  I wherein: m is an integer greater than 1; (cOpR)is a coated optical reporter particle, wherein the cOpR comprises anupconverting phosphor (UCP) material of the formulaNa(Y_(x)Yb_(y)Er_(z))F₄, and wherein: x is from about 0.6 to about 0.95;y is from about 0.05 to about 0.35; and z is from about 0.1 to about0.001; (NA) is a nucleic acid oligomer; and L is a linking groupcovalently bound to the coated optical reporter particle and to thenucleic acid oligomer, wherein the linking group L comprises an alkylenemoiety having a first end covalently bound to the coated opticalreporter particle and a second end covalently bound to the nucleic acidoligomer.